The biochemical mechanisms of protein degradation, characterized by an ATP dependence, have until recently been completely obscure. We have recently resolved a fully ATP-dependent cell-free system from rabbit reticulocytes into several complementing activities, all of which are absolutely required to reconstitute the entire system. This resolution now enables us to explore, by direct biochemical methods, the intermediary reactions of what now seems to be a complex process and its control. In particular, the novel and unusual role of ATP in this catabolic pathway can now be closely examined. In the present research, we shall use purified enzyme components and protein factors from reticulocytes to distinguish between alternative possible mechanisms of ATP action. These are: chemical modification of an enzyme or an activator, phosphorylation of the polypeptide substrate, a coupling mechanism between ATP hydrolysis and peptide bond cleavage, or an allosteric activation by ATP. The possible association of the individual components into an active multi-enzyme complex, as well as the role of ATP in the assembly or organization of such a structure, will also be investigated. We will examine similar ATP-dependent cell-free proteolytic systems in other tissues subject to physiological regulation, such as the heart, skeletal muscle or the liver. These cell-free systems will then be utilized to explore the regulatory mechanisms involved in the marked effects of insulin or growth-promoting agents, on protein degradation. Thus, this study may not only yield important information on the novel biochemical mechanisms of protein degradation, but may also shed light on the regulatory mechanisms of this fundamental process.